Field of the Invention
The present invention relates to a system for confocal observation of a sample.
Description of Related Art
Spinning disc systems (i.e., confocal microscope systems based on the Nipkow principle), comprising camera-based parallel detection, have become increasingly popular in live cell microscopy. The method for imaging using a plurality of light spots was first described by P. G. Nipkow in 1884 and was used subsequently, for example, in (mechanical) television technology (“Televisor”). Therein a pinhole disc is rotated around a central rotation axis, and all areas of the object to be observed are illuminated through the pinholes equally long. The rotating disc is generally called Nipkow disc. Nipkow microscope systems are fast and gentle to live cells; however, they suffer from a low light efficiency if only the small fraction of light is utilized in the excitation beam-path which passes through the pinholes without prior focusing.
In the 1990's Japanese engineers at the Yokogawa company were first to successfully utilize micro-lenses in a corresponding second disk to concentrate excitation intensity onto the pinholes of the first disk, thus increase the light throughput considerably. To this end the second disc is located in the illumination beam path in front of the pinhole disc in such a manner that the pinhole plane coincides with the focal plane of the micro-lenses and that the focal points generated by the micro-lenses fall exactly onto the pinhole openings. If now both discs rotate around a common axis, one obtains many (typically more than 1000) simultaneously illuminated spots on the sample which—by rotation—sequentially illuminate the entire area of the sample seen by the detector. The emission generated by these illuminated spots is then detected by a camera with confocal filtering through the pinholes. Examples for such methods, wherein both the excitation light and the emission light passes through the pinholes, are described in U.S. Pat. Nos. 5,428,475, 5,717,519 and 6,300,618.
There are substantial drawbacks of methods where both excitation-and emission beams have to pass through the same pinholes: They result from the combination and separation, respectively, of the excitation beam and the emission beam, which has to take place in the finite optical space between the micro-lenses disc and the pinhole disc. These inherent drawbacks can be avoided if—as described in U.S. Pat. No. 7,706,043 B2 or 7,580,171 B2—combination and separation of excitation beam path and emission beam path, respectively, are realized in a real infinite space which is located, as seen from the objective, behind the Nipkow disc. In the examples described in U.S. Pat. No. 7,706,043 B2 or 7,580,171 B2 micro-optics and pinholes are combined in a common disc, wherein the excitation light and the emission light cross the same pinholes, but in opposite direction.
For the case of excitation by multi-photon absorption A. Egner and S. W. Hell have presented in “Time multiplexing and parallelization in multi-focal multi-photon microscopy”, J. Opt. Soc. Am. 17, 1192-1200, in 2000 a two-photon spinning disc system wherein spatial filtering of the emission light through confocal pinholes—due to the two-photon excitation—is not mandatory. In the same year, K. Fujita at al. (Optics Communications, vol. 174, pages 7-12) have shown that with the help of pinholes image quality may be improved.